Site selectively tagged and templated molecularly imprinted polymers for sensor applications

ABSTRACT

This invention provides molecularly imprinted polymers (MIPs) for the detection of analytes, methods for forming the MIPs and detecting the analyte using the MIPs. The MIP comprises templated sites which are formed using a mimic of the analyte such that a reporter compound can be selectively attached at the templated sites, thus providing a site selectively tagged and templated MIP.

This application claims the priority of U.S. Provisional application No.60/527,661 filed on Dec. 8, 2003, the disclosure of which isincorporated herein by reference.

This work was funded by Grant No. CHE-0078101 and CHE-0315129 from theNational Science Foundation. The Government has certain rights in theinvention.

FIELD OF THE INVENTION

This invention relates to field of detection of analytes by the use ofselectively tagged and templated molecularly imprinted polymers(SSTT-MIPs).

BACKGROUND OF THE INVENTION

Americans spend billions of dollars annually on the detection andquantification of chemical substances. Most of these measurements areperformed in well-outfitted laboratories, requiring skilled personnel,large amounts of costly reagents, and long analysis times. Also, thedemands for use in clinical point-of-care testing or for fielddeployment necessitate small, integrated analytical platforms. Many ofthese needs have helped to spark chemical sensor development [1].Similarly, the ever growing need to simultaneously measure “everything”in a sample [2] has pushed the development of artificial “noses” and“tongues” [3] which depend upon chemical and biochemical sensor arraystrategies [4-10]

Presently, there is a need to develop new devices which overcome thedisadvantages of presently used methods. Detection methods which allowthe simultaneous quantification of multiple analytes in a sample, areless expensive and more simple to construct and operate, are accurate,precise and reliable, and provide adequate detection limits andselectivity would be a welcome advance in the field of analytedetection.

On general method which has been tried is the use of“biosensors.” In thegeneric biosensor, an immobilized biorecognition element (e.g., anantibody, aptamer, DNA oligonucleotide, enzyme, lectin, signalingprotein, transport protein) serves to selectively recognize a targetanalyte and the binding or conversion (if the analyte is a substrate)event leads to an optical, mass, thermal, or electrochemical responsethat is related to the analyte concentration within the sample.

Although biosensor development may appear simple, there are manyfundamental issues associated with developing analytically usefulbiosensors. For example, traditional strategies depend upon identifyingan appropriate biorecognition element that can selectively recognize thetarget analyte. A suitable detection/transduction method is used and thebiorecognition element is immobilized [11-13] such that it retains itsnative activity/affinity and selectivity. The biorecognition element—thebiosensor's heart in a traditional design—needs to remain stable overtime, the target analyte needs to have access to the biorecognitionelement, and the analyte-biorecognition element association/interactionneeds to be reversible or at least easily dissociated/reset followingeach measurement. The foregoing shortcomings have limited theapplication of biosensors in analyte detection.

Over the past decade, the introduction of specific binding domainswithin synthetic polymers by template-directed cross-linking offunctional monomers has attracted considerable attention [15]. Molecularimprinting involves arranging polymerizable functional monomers around atemplate (pseudo-target analyte or the actual target analyte) followedby polymerization and template removal.

Arrangement is typically achieved by: (i) non-covalent interactions(e.g., H-bonds, ion pair interactions) or (ii) reversible covalentinteractions. After template removal, these molecularly imprintedpolymers (MIP) can recognize and bind specific chemical species (i.e.,the template or template analogs).

Potential advantages of MIP-based materials include: specificitycomparable to a biorecognition element; robustness and stability underextreme chemical and physical conditions; and an ability to designrecognition sites for analytes that lack suitable biorecognitionelements. MIPs have been developed for (not an exhaustive list)proteins, amino acid derivatives, sugars and their derivatives,vitamins, nucleotide bases, pesticides, pharmaceuticals, and polycyclicaromatic hydrocarbons. However, according to Lam [16], one of the majorissues in the development of MIP based biomimetic sensors is signaltransduction.

There are several reports of MIP based sensors that exploit luminescenceas the transduction modality. For example, the Powell group [17a] formedcAMP-imprinted organic polymers by usingtrans-4-[p-(N,N-dimethylamino)stryl]-N-vinylbenzylpyrimidinium chloride(fluorophore), trimethylolpropane trimethacrylate, 2-hydroxyethylmethacrylate, and the initiator, 2,2′-azobisisobutyronitrile (AIBN).These MIPs showed a 20% change in fluorescence in the presence of 1millimolar cAMP and they were selective for cAMP over cGMP. The Murraygroup [17b] prepared Soman-imprinted organic polymers by usingEu(R)₃(NO₃)₃ (R=pinacolyl methylphosphonate or divinylmethyl benzoate)(fluorophore), styrene, and AIBN. These MIPs were able to detect Somandown to 750 parts per quadrillion and interferences fromorganophosphorous pesticides was minimal. The sensor response time was 8min. The Takeuchi group [17c] reported a fluorescence-based MIP sensorfor the detection of 9-ethyladenine (9-EA). This sensor was based ontemplating 9-EA with5,10,15-tris(4-isopropylphenyl)-20-(4-metharcyloyloxyphenyl)porphryinzinc (II) (fluorophore) and methacrylic acid. In CH₂Cl₂, these polymersexhibited a 9-EA binding affinity of 7.5×10⁵ M⁻¹, were selective overadenine, 4-aminopyridine, and 2-aminopyridine, and yielded afluorescence change of 40% in the presence of 250 micromolar 9-EA. TheWang group [17d] reported on a fluorescence-based MIP sensor fordetecting L-tryptophan that used a dansylated dimethylacrylic acidmonomer (fluorophore), ethyleneglycol dimethylacrylate, and AIBN. Inoperation the authors loaded a mobile quencher, 4-nitrobenzaldehyde(4-NB), into the MIP which quenched the dansyl emission. Upon additionof L-tryptophan some of the 4-NB was liberated/blocked from accessingthe dansyl residue and the dansyl fluorescence increased. The change influorescence upon adding 10 millimolar L-tryptophan was 45%. Thepresence of an equivalent amount of D-tryptophan, L-phenylalanine, andL-alanine caused 32%, 27%, and <9% changes in fluorescence. The Lamgroup [16] used a photoinduced electron transfer (PET) strategy to forma fluorescence-based MIP for the detection of 2,4-dichlorophenoxyaceticacid (2,4-D) within a templated sol-gel-derived xerogel. In this work,the authors copolymerized 3-[N,N-bis(9-anthrylmethyl)amino)]propyltriethoxysilane (fluorophore) with tetraethoxysilane (TEOS) andphenyltrimethoxysilane (PtrMES) using 2,4-D as the template. The soformed MIP exhibited a change in fluorescence with pH (apparent pKa near7.2) and it yielded a 15% decrease in fluorescence in the presence of750 micromolar 2,4-D. Tests with benzoic acid and acetic acid at similarconcentrations did not cause significant interference.

Most recently, Edmiston and coworkers [17e] reported an approach tofabricate a fluorescence-based xerogel MIP for the detection of thepesticide 1,1-bis(4-chlorophenyl)2,2,2-trichloroethane (DDT) by using asacrificial spacer (SS) scheme [18] wherein they reacted3-isocyanatopropyltriethyoxysilane with 4,4′-ethylidenebisphenol to formthe SS. They then prepared the fluorescent monomer by reacting3-aminoproplytriethoxysilane (APTES) with the fluorophore4-chloro-7-nitrobenzofurazan (NBD) (attaching the NBD to the APTESamine, NBD-APTES). The imprinted xerogel was then formed by mixingNBD-APTES, SS, and bis(trimethoxysilyl)benzene followed by a typicalacid hydrolysis protocol. Once the xerogel was formed, the authorscleaved the SS carbamate bond with dilute LiAlH₄ to form amine residueswithin the template site, and liberating the SS from the xerogel. Thesensor responded to DDT (3% change in NBD fluorescence) and thetemplated xerogels offered selectivity for DDT over potentialinterferents (e.g., anthracene (A),2,2-bis(4-chlorophenyl)-1,1-dichloroethylene (p,p-DDE),1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,2-dichloroethane(o,p-DDD),2,2-bis(4-chlorophenyl)-1,1-dichloroethane (p,p-DDD),diphenylmethane(DPM), 4,4′-dibromobiphenyl (DBBP), 4,4′-bis(chloromethyl)-1,1′-biphenyl(BCP)). The DDT detection limits were at the single digit part perbillion level.

However, in all previous work on luminescence based MIP sensors, nostrategy has been developed to ensure that the luminescent reportermolecule is actually in immediate proximity to the analyte when theanalyte binding occurs.

SUMMARY OF THE INVENTION

The present invention presents a method for forming a reliable chemicalsensor platform based on site selectively tagged and templatedmolecularly imprinted polymers. The SSTT-MIP strategy used in thepresent method provides a way to form a MIP having a templated sitespecific for an analyte and at which a reporter molecule can also beattached. In this way, analyte detection can be carried out with ahigher efficiency in comparison to methodologies without any provisionfor such positioning. With this invention, measurement characteristicssuch as signal-to-background and signal-to-noise ratios are expected tobe improved over those of similar MIP methods such as those described byEdmiston.

The method comprises the following steps. A mimic of the analyte, termedherein as the sacrificial spacer molecule template (SSMT), is identifiedor created. Next, the SSMT is incorporated into a polymer platform byinitiating polymerization of the unpolymerized components in thepresence of the SSMT resulting in the formation of polymer platformshaving templated sites at which the SSMT is bonded by a plurality ofreactive groups. The SSMT is then removed from the polymer platform,creating within the platform one or more templated sites for the targetanalyte and one or more reporter molecules. The SSMT is chosen such thatremoval of the SSMT leaves reactive groups for the binding of theanalyte and at least one reporter molecule at the template site.Following removal of the SSMT, the template site is contacted with theanalyte which bonds to it thereby blocking all reactive groups exceptfor the group to which reporter binding (i.e., tagging) is to takeplace. This complex is then exposed to the reporter molecule. Theanalyte is removed, and the resulting polymer platform is asite-selectively tagged and templated molecularly imprinted polymer(SSTT-MIP).

The present invention also provides molecularly imprinted polymerplatforms in which templated sites are formed for specific analytes. Inone embodiment, the polymer platforms can be provided wherein thetemplated sites have at least one reporter molecule bonded to a reactivegroup at the site. In one embodiment, the polymer platform comprisesxerogels or aerogels.

This method can be used to develop sensors for the detection of a widevariety of analytes including those for which no known biologicalrecognition molecules have been reported.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a representation of a typical reaction scheme to produce asite-selectively tagged and templated molecularly imprinted polymer(SSTT-MIP) based on a sol-gel-derived xerogel platform. The targetanalyte is DDT.

FIG. 2 is a representation of a binary templating strategy for forming adeoxynucleoside selective SSTT-MIP sensor element.

FIG. 3 is a representation of a response profile for a SSTT-MIP sensorthat was designed (see FIG. 1) for the detection of DDT. HBHE is1-hydroxy-2,2-bis(4-hydroxyphenyl)ethane; a hydroxylated analog of DDT.

FIG. 4 is a representation of the response from three different SSTT-MIPsensors, each templated for DDT, when challenged with 100 ppb of theindicated molecules. The three SSTT-MIP sensors were derived fromdifferent precursors having different R′ residues (a non-exhaustivelisting of example R′ residues are defined in the inset and FIG. 1).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides site selectively tagged and templatedmolecularly imprinted polymers (SSTT-MIP). The SSTT-MIPs have a reportermolecule strategically placed to enhance the efficiency of detection ofthe analyte. While not intending to be bound by theory, it is thoughtthat changes in the physicochemical properties (e.g., dielectricconstant, refractive index, dynamics, etc.) of the immediatemicroenvironment that surrounds the reporter molecules (referred toherein as a reporter's cybotactic region) cause changes in the reportermolecule's absorbance, excitation and emission spectra, excited-stateluminescence lifetime and/or luminescence polarization. As a result, agreater change in reporter absorbance/luminescence properties (i.e.,analytical signal) is expected to be realized when a reporter moleculeand the MIP template site share some or all of the reporter molecule'scybotactic region. Hence, when analyte molecules are bound to a MIPtemplate site thereby changing the physicochemical properties of the MIPtemplate site, the binding is sensed simultaneously by the reportermolecule at the MIP template site.

In one embodiment of the invention, reporter molecules are exclusivelypresent at the template sites, thereby reducing the signal to noiseratio and signal to background ratio. In this embodiment, very few or noreporter molecules are present in the polymer matrix (i.e., other thanat the templated sites).

The present invention also provides a method for fabricating a MIP inwhich a reporter molecule can be selectively installed at a templatedsite such that during the detection of the analyte, the reportermolecule is positioned in close proximity to the analyte (i.e, withinthe reporter molecules cybotactic region).

As used herein, the term “molecularly imprinted polymer” or “MIP” refersgenerally to a gel or polymeric mold-like structure having multiplemolecular-scale templated sites, each of which has the ability tospecifically bind an analyte molecule. The templated sites complementthe shape of at least a portion of an analyte molecule. Furthermore, thetemplated sites contain interactive moieties that have a positioningwhich matches the spatial arrangement of moieties on the analyte. Themoieties in the templated site are complementary to the moieties on theanalyte. By complementary, it is meant that moiety in the templated sitecan interact with the positionally corresponding moiety on the analyteto form a bond or other attractive association, such as, for example, anelectrostatic, covalent, ionic, hydrophobic, hydrogen-bonding, or otherinteraction.

As will be recognized by those of skill in the art, MIPs are typicallyformed by mixing analyte molecules with one or more functional monomersto form imprint/monomer complexes (wherein the imprint moleculeinteracts or bonds with a complementary moiety of the functional monomervia covalent, ionic, hydrophobic, hydrogen-bonding, or otherinteractions). The monomer/analyte complexes are then polymerized into acrosslinked polymer matrix. The analyte molecules are subsequentlydissociated/cleaved from the functional monomers and thereby removedfrom the polymer matrix to leave templated recognition sites. Thetemplated sites have a shape which is specific to the analyte. Themolecular-scale cavities contain moieties which are complementary to theanalyte molecule, giving the cavity the ability to selectively bind theanalyte.

In contrast to MIP methods of the prior art, in the method of thepresent invention, a mimic of the analyte, termed herein as thesacrificial spacer molecule template (SSMT) is identified or created,and then incorporated within a polymer platform. The SSMT preferablybears close structural resemblance to the target analyte. Furthermore,the SSMT is chosen such that it forms bonds with the polymer platform inexcess of the number of those which will be formed by the bound analyte.The SSMT should have at least one additional reactive group so as to beable to bind to the polymer matrix at an additional site relative to anintended analyte. In one embodiment, the number of reactive groups onthe SSMT which link it to the polymer platform, is at least one morethan the number of reactive groups on the corresponding analyte. In afurther embodiment, the SSMT is structurally identical to the analyteexcept for the number and identity of the reactive groups which link tothe polymer platform. The groups which participate in the SSMT-polymerlinkages may be included in the SSMT. These SSMT polymer linkages areultimately cleaved in the removal of the SSMT.

In general, analytes and the corresponding SSMT are structurallysimilar, often differing by the presence of an additional functionalgroup on the SSMT. However, chemically different molecules may besimilarly shaped, and thus, close structural similarity may not be acomplete characterization of the relationship between analyte and SSMT.Furthermore, there are many slight structural modifications which maygive rise to acceptable SSMTs for a given analyte. Other effects, suchas intermolecular hydrogen bonding, ionic interactions, aggregation,encapsulation, dimerization, may still result in an SSTT-MIP whichexhibits a binding preference toward the analyte molecule.

The removal of the SSMT results in templated sites which are referred toherein for the sake of simplicity as cavities, within the polymerplatform. Exposed at each templated site are reactive groups which areresponsible for target analyte recognition. However, as a consequence ofthe additional reactive group(s) mentioned above, when the SSMT iscleaved from the cavity, the cavity bears one or more reactive groups inexcess of the groups needed to bind an analyte molecule. The extragroup(s) is (are) used to bond with reporter molecules. Once thereporter molecule(s) is (are) bound at the templated site, theabsorbance/luminescence from the SSTT-MIP is measured and a change inUV, visible or IR absorbance/luminescence properties of the reporter(e.g., absorbance spectra, excitation and emission spectra,excited-state luminescence lifetime and/or luminescence polarization)indicates the presence of an analyte molecule at the templated site. Thetotal change in absorbance/luminescence is generally proportional to theconcentration of analyte molecule in the sample. Thus, analyte-dependentcalibration curves can readily be constructed.

In one embodiment, the SSMT, in comparison to its target analyte,possesses one additional functional residue, as illustrated further inFIG. 1 in the case of the analyte DDT and an appropriate SSMT molecule,Compound 1. Compound 1 is an analog of DDT with four (EtO)₃-Si-tippedcarbamate-containing groups. DDT, in contrast, has three Cl-containingfunctional groups in positions which are analogous to three of thepositions of the above-mentioned groups on Compound 1. A MIP is createdby conducting a polymerization with one or more functional monomerswhich binds to the Compound 1 through the available alkoxide functionalgroups. The polymerization occurs around the SSMT, giving a polymerplatform which is linked to the SSMT via the (EtO)₃-Si carbamate groups.The SSMT is then cleaved from the polymerized platform to give a MIPwith templated sites, each site having four amino groups extending intoit. The MIP is then exposed to DDT, which binds into the template sitewith an affinity which depends, in part, on the composition of thepolymer and its preparation protocol. The DDT molecules bind to thetemplate site by also interacting with three of the four amino groups inthe template site (i.e., each of the three Cl residues on DDT hydrogenbonds to an —NH2 residue within the template site). Because of the“extra” —OH residue on Compound 1, when it is cleaved from the templatedcavity using the foregoing step, an amine residue in addition to thoseneeded to bind the analyte, DDT, is formed at the template site. Thus,within the template site there is a single free amine residue that isnot bound to the analyte, DDT. The free amine residue is also notblocked by the presence of the analyte, DDT. This free amine residuewithin the template site is then site-selectively tagged with a reportermolecule, for example, Lissamine™ or rhodamine B sulfonyl chloride, toplace a reporter molecule such that its cybotactic region largelyoverlaps the templated site where analyte will bind. The analyte and anyunreacted luminescent probe are then washed from the polymerizedplatform to produce the SSTT-MIP. The use of a hydroxyl residue andcarbamate chemistry is an illustrative example; and one skilled in theart will recognize other strategies. Preferred are chemistries which canbe used without appreciably affecting the integrity of the polymerplatform, with the use of carbamate linkages being most preferred.

It should be noted that after dissociation from the SSTT-MIP, the SSMTmay be modified. For instance, in the example of FIG. 1, the nitrogenatom from each carbamate linkage is left behind as an amine residue whenthe linkage is cleaved. The modified SSMT molecule is termed herein asSSMT′ (SSMT prime).

The present invention can be used with chemistries in which the bondsbetween the functional monomers and SSMT, created before polymerization,are dissociated/cleaved upon release of the SSMT. Such a situation couldarise for example, in the case of a hydroxyl-containing functionalmonomer which forms an ester linkage with a carboxylicacid-containing-SSMT, which is cleaved upon release of the SSMT from theSSTT-MIP formed from polymerized functional monomer. However, it ispreferred to import the entire linkage to be cleaved (carbamate as inthe above example) with the SSMT (as done in FIG. 1). In such cases, theSSMT′ is characterized as missing a portion of the linkage which remainsattached to the cavity in order to bind with the analyte.

Many different types of polymer systems can be used in the method of thepresent invention. As an illustrative example, a sol-gel derived xerogelcan be used. However, the approach can easily be adapted to other MIPsbased on aerogels or natural or synthetic polymer systems. However,sol-gel-derived xerogels and aerogels are convenient because thexerogels are nanoporous materials with physicochemical properties andpore sizes that can be tuned by ones choice of precursor(s) and theprocessing protocol such as described in references 19-28.

In general, the polymer used in the method of this invention should besuch that the chosen SSMT can bind to or otherwise interact chemicallyto at least some of its component momomers prior to polymerization.Thus, for example, an SSMT which has linkage groups which containingterminal (EtO)₃—Si— groups, such as the (EtO)₃—Si-carbamate groups shownin FIG. 1, can be used with a polymerization system which containspolymerization precursors which can form the siloxane by hydrolyzingwith this alkoxide-based SSMT. Non-limiting examples of acceptablepolymerization precursors are, as shown in the figure,(EtO)₃—Si—R′—Si-(EtO)₃ and (EtO)₃—Si—R″ groups. Other examples are knownto those skilled in the art (cf., references 19-28).

For sacrificial spacer molecule templates (SSMTs) that contain multiplereactive sites, these additional sites can be blocked (protected) byusing protecting groups. Examples of such protecting groups arebenzyloxycarbonyl, t-butoxycarbonyl (t-BOC), 9-fluorenylmethoxycarbonyl(Fmoc), or phenyl-SO₂Cl). Such protection/deprotection is known to thoseskilled in the art and is described in reference 29.

In general, the SSTT MIP method is particularly suited for analytes forwhich a suitable SSMT analog having one or more extra functional groupswith respect to the analyte can be prepared.

The SSTT-MIP should be able to associate or bind with the analyte. Forinstance, the association shown in FIG. 1 is an example of (hydrogenbonding as well as hydrophobic and π-π interactions). Other types ofassociations are hydrophobic, hydrogen-bonding, etc. and combination ofinteractions that exploit specific chemical aspects of the analyte andthe polymer-based platform.

The reporter molecule in the present invention may be a luminophore or achromophore. A binding group can be attached to the reporter molecule orin some cases, the reporter molecule may already have a binding groupattached. In the preparation of the SSTT-MIPs, the reporter moleculescan be directly bonded to the template site via the binding group orindirectly by using a connecting moiety between the binding group andthe luminophore/chromophore. In one embodiment, the reporter is aluminophore connected to a binding group via a connecting group. Bindinggroups are commercially available (such as from Molecular Probes). Anon-limiting example of a reporter comprised of a luminophore, aconnecting moiety, and a —SO₂Cl binding group is illustratedspecifically in FIGS. 1 and 2.

The luminophore could be an organic or inorganic species. Examples ofluminophores include organic species like fluorescein, BODIPY,rhodamine, organometallic complexes liketris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) ([Ru(dpp)₃]²⁺ andluminescent nanoparticles (i.e., quantum dots). The quantum dots mayhave elemental silicone (Si), cadmium sulfide (CdS) and zinc selenite(ZnSe)

The connecting moiety (also referred to herein as a tether) can be oneof any possible natural or synthetic groups that have been used to spaceresidues apart from one another in the chemical sciences. Generalexamples of connecting moieties are methylene chains, ether chains,polydimethylsiloxane chains, polystyrene chains, amino acid chains, andany other organic/inorganic oligomer.

Specific examples of binding groups which can be used to form linkageswith specific types of groups attached to the templated site are asfollows: to tag/label amine residues one can use isothiocyanates,succinimidyl esters, carboxylic esters, tetrafluorophenyl esters,carbonyl azides, sulfonyl chlorides, arylating agents and aldehydes; totag/label thiol residues one can use iodoacetamides, maleimides, alkylhalides, arylating agents, and disulfides; to tag/label alcohol residuesone can use dichlorotriazines, N-methylisatoic anhydride,aminophenylboronic acids, isocyanates prepared from acyl azides, andacyl nitriles; and to tag/label carboxylic acids one can use hydrazines,hydroxylamines amines, carbodiimides, esterification reagents,diazoalkanes, alkyl halides, and trifluoromethanesulfonates.

It is preferred that the connecting moiety not be so long as to removethe chromophore/luminophore from the template site to such a degree thatthe effect on the chromophore/luminophore of the analyte moleculebinding to the template site is too small to detect. Accordingly, someoverlap between the reporter molecule's cybotactic region and thetemplate site is desirable. Alternatively, the reporter molecule can bedirectly attached to one of the aforementioned binding groups.

The reporter molecule generally has a luminescence spectrum in the UV,visible and/or IR; however, non-luminescent dye molecules that areresponsive to their physicochemical environments can also be used asreporter molecules (e.g., 4-nitroaniline, and2,6-diphenyl-4-(2,4,6-triphenyl-1-pyridinio)phenolate (Reichardt's dye30), 2,6-dichloro-4-(2,4,6-triphenyl-1-pyridinio)phenolate (Reichardt'sdye 33), and N,N-diethyl-4-nitroaniline) in the SSTT-MIP strategy. Thechromophore's/luminophore's absorbance and/or emission depends on thephysicochemical properties of its local environment such that the molarabsorbance, absorbance spectrum, emission quantum yield, emissionspectrum, excited-state lifetime, and/or luminescence polarization are(one or all) modulated to some degree by the local physicochemicalproperties that surround the reporter molecule. This change inphysicochemical properties is modulated by the binding of the analytemolecule to the template site and is detected as a change in thereporter molecule's molar absorbance, absorbance spectrum, emissionquantum yield, emission spectrum, excited-state lifetime, and/orluminescence polarization. Examples of suitable luminophores includedansyl, fluorescein, rhodamine, NBD, andtris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) ([Ru(dpp)₃]²⁺.Examples of suitable chromophores include2,6-diphenyl-4-(2,4,6-triphenyl-1-pyridinio)phenolate (Reichardt's dye30), 2,6-dichloro-4-(2,4,6-triphenyl-l -pyridinio)phenolate (Reichardt'sdye 33), and N,N-diethyl-4-nitroaniline.

Below are examples of analytes that can be detected by the presentSSTT-MIP methods outlined in FIGS. 1 and 2. The possible analytemolecules include, but are not limited to, pharmaceuticals, steroids,prostaglandins, glycosides, deoxyribonucleosides, deoxyribonucleotidesand the like. A non-limiting list of analytes that can be detected bythis method and their sacrificial spacer molecule template are shown inTable 1. TABLE 1 Examples of sensor target analytes (left column) andSSMT (right column). Pharmaceutical Pheytoin 5-(p-Hydroxyphenyl)-5-phenylhydantoin Codeine Morphine Heroin 3-Acetylmorphine Cocainep-Hydroxycocaine Δ9-THC 8-α-Hydroxy-Δ9-THC Tetracycline OxytetracyclineMorphine Nalbuphine Steroids Testosterone 4-HydroxytestosteroneEstradiol 4-Hydroxyestradiol Progesterone 17-α-HydroxyprogesteroneCholesterol 22(r)-Hydroxycholesterol Prostaglandins Prostaglandin A219(R)-Hydroxyprostaglandin A2 Prostaglandin B219(R)-Hydroxyprostaglandin B2 Prostaglandin E119(R)-Hydroxyprostaglandin E1 Prostaglandin E219(R)-Hydroxyprostaglandin E2 Prostaglandin F1a19(R)-Hydroxyprostaglandin F1a Prostaglandin F2a19(R)-Hydroxyprostaglandin F2a Prostaglandin F2a19(R)-Hydroxyprostaglandin F2a Glycosides Methyl α-D-glucopyrnosideα-D-glucopyranose Methyl β-D-glucopyrnoside β-D-glucopyranose DigitoxinDigoxin 2-Deoxyribose Ribose

The SSTT-MIP strategy is particularly effective in the preparation ofsensors to detect and quantify pharmaceuticals (e.g., phenytoin,codeine, heroin), prostaglandins (e.g., A2, B2, F1a), steroids(testosterone, estradiol, progesterone), and glycosides (methyl∀-D-glucopyranoside, digitoxin, 2-deoxyribose). However, the method canbe used to prepare sensors for the detection of a wide range ofanalytes.

For the detection of deoxyribonucleotides, a binary templating strategycan be used as shown in FIG. 2 to form the SSTT-MIPs. The base ribosesacrificial spacer (Base-Ribose-SSMT) is formed by using the carbamateformation chemistry illustrated in FIG. 1. The base residue on theBase-ribose-SSMT can be selected to match the base on the target analyteexcept that uracil (U) is used to template thymine (T). The amine siteson the bases can be protected as needed using the Fmocprotection/deprotection to prevent urea formation. The Base-Ribose-SSMTcan be mixed (1:1) with the appropriate secondary base template(Base-2°-SSMT) and the main component of the xerogel (i.e.,(EtO)₃Si—R—″Si(EtO)₃, Si(EtO)₃, and/or (EtO)₄Si)), the templated xerogelformed, the sacrificial spacer removed, and the template site issite-selectively tagged with a chromophore/luminophore. The base residueon the Base-2°-SSMT can be selected to complement the base on the target20 analyte (such as A for T and G for C and vice versa). TheBase-2°-SSMT can be prepared in two steps. As an example, Leonard'smethod [30] can be used to produce 9-(3-chloropropyl)adenine,9-(3-chloropropyl)guanine, 3-(3-chloropropyl)thymine, and3-(3-chloropropyl)cytosine. The derivitized bases are then reacted with3-mercaptopropyl-triethyoxysilane (MPTES) to form Base-2°-SSMT shown inFIG. 2.

In one embodiment of the invention, multiple tunable sensors can bedesigned and developed for each target analyte. Here, simultaneousscreening of SSTT-MIP libraries can be performed to optimize SSTT-MIPanalytical performance and identify sets of SSTT-MIPs wherein theresponse characteristics, within the set, exhibit the greatest diversityfor a given analyte. In one illustration of this (FIG. 3) response dataare presented to demonstrate that SSTT-MIPs can yield substantialdifferences in response for one analyte over another. A second exampleis presented in FIG. 4 showing the significant changes in response froman SSTT-MIP platform are possible simply by changing one of theprecursors that is used to form the xerogel from(EtO)₃—Si—(CH₂)₂—Si—(OEt)₃ to (EtO)₃—Si-phenyl-Si—(OEt)₃ to(EtO)₃-Si-biphenyl-Si—(OEt)₃ to create three different xerogel-basedSSTT-MIPs.

By using such multiple sensors in concert, the overall detectionaccuracy, precision, and dynamic range can be improved. False positivesand negatives may also be more readily detected by using multipletunable sensors and redundant detection schema.

Arrays of discrete SSTT-MIP-based sensor elements can be formed on theface of a light source and detected with an array-based detector.Formation of sensor arrays on the face of a light emitting diode (LED)and the simultaneous detection of multiple analytes are described inU.S. Pat. Nos. 6,492,182, 6,582,966 and 6,589,438 incorporated herein byreference. Each SSTT-MIP-based sensor element can serve as an individualsensor for a particular target analyte. In operation, the LED serves asthe light source to simultaneously excite the chromophores/luminophoreswithin all the SSTT-MIP sensor elements on the LED face and the targetanalyte-dependent absorbance/emission from all the SSTT-MIP-based sensorelement can be detected by an array detector (e.g., charge coupleddevice (CCD), complementary metal oxide semiconductor (CMOS)). Thereporter molecule absorbance/luminescence from each SSTT-MIP-basedsensor element is then related to the corresponding target analyteconcentration in the sample.

The absorbance/luminescence from an SSTT-MIP sensor array can bedetected with a wide variety of photonic detectors. Examples of photonicdetectors include a photodiode, photomultiplier tube, charge coupleddevice (CCD) or CMOS based image detector. When the detection device isan array detector, an entire SSTT-MIP array of sensor elements can beevaluated simultaneously.

In another embodiment, pin printing methodologies can be used to developsensor arrays for simultaneous multi-analyte detection. This allows ahigh-speed imprinting of a plurality of sensor elements on a substratethat can be excited by an extrinsic light source (laser, lamp) or on theface of a LED for direct, integrated excitation/sensing.

The invention is further described in the Examples presented below whichare meant as illustrative and not intended to be restrictive in any way.

EXAMPLE 1

This embodiment describes the preparation of a SSTT-MIP for theselective detection of DDT as the target analyte (FIG. 1). First, asacrificial spacer molecule template (SSMT) was synthesized. For thisexample, this was a two-step process. In step1,4,4′,4″-trihydroxytriphenylmethanol (HBHE) was prepared by using thesynthetic strategy reported by Cushman [31]. In step two, the completeSSMT was formed by reacting 1 eq of 4,4′,4″-trihydroxytriphenylmethanolwith an excess of 3-isocyanatopropyltriethyoxysilane (IPTES) in dry THFas described to form the tetracarbamate, (Compound 1) the SSMT. ¹H and¹³C NMR (500 MHz), IR, and MS data (not shown) were all consistent withthe formation of all four carbamate bonds in Compound 1. The templatedxerogel was then prepared by reacting 1 eq of Compound 1 with 100-500 eqof one of several alkoxides (see FIG. 1). This mixture was agitated forseveral minutes to several hours. After allowing the sol to hydrolyze ina sealed vial, thin films (500-800 nm, determined by profilometry) werespun cast onto a fused silica substrate and the xerogel was allowed toform in the dark, for 24-48 hours, at room temperature. The SSMT wasremoved from the templated xerogel by using 50-200 mM LiAlH₄ [17e]followed by a sequential rinse with THF, 0.1 M HCl, and 0.1 M NH₃. Toinstall the reporter molecule into the templated site we contacted thetemplated xerogel films with 10-200 ppm DDT in THF to fill accessibletemplated sites with DDT. This left a single free amine residue (FIG. 1)within each template site. The reporter molecule was then introducedinto the THF solution with a micropipette and the reaction between thefree amine and the binding group in the reporter (—SO₂Cl) was allowed toproceed in the dark at room temperature for 2 hours. The xerogel filmwas then rinsed with fresh THF to remove any unreacted reporter moleculeand DDT to create the SSTT-MIP.

EXAMPLE 2

This example describes the use of SSMT-MIPs prepared as described inExample 1. FIG. 3 summarizes the response profiles from a series ofxerogel-based SSTT-MIP films that were designed for the detection ofDDT. The detector for these particular experiments was a photomultipliertube. For these particular SSTT-MIPs, biphenyl was used as the R′residue (see FIG. 1). When DDT was added to the SSTT-MIP thefluorescence decreased (quenching) in step with the addition of DDT. Asan initial test of the SSTT-MIP's selectivity for DDT these SSTT-MIPfilms in THF were reacted with an excess of phenyl-SO₂Cl to form asulfonamide with any free amines. The response of the phenyl-SO₂Cltreated SSTT-MIP films was then re-determined. There was no observableresponse over the DDT concentration range tested. This result suggeststhat the phenyl-SO₂Cl accessed the templated sites in the SSTT-MIP inthe absence of DDT and formed the sulfonamide with one or more of thefree amine residues within the accessible DDT template site within theSSTT-MIP. In a second test, HBHE was introduced to SSTT-MIPs that weretemplated for DDT. HBHE, a hydroxylated DDT analog, was readilyrecognized by the SSTT-MIP; however, the observed response profile wascompletely different in comparison to DDT response. This resultdemonstrates that analyte-dependent response profiles are possible withthe SSTT-MIPs. Again, when these SSTT-MIP films were treated withphenyl-SO₂Cl, there was no response to HBHE (results not shown).However, when a SSTT-MIP that was templated for DDT was initiallychallenged with a THF solution that contained 80 ppb HBHE, followed bythe addition of 250 ppb DDT (waiting 15 min), the luminescence initiallyincreased by 125±3% in the presence of HBHE and then decreased by 9±2%when the DDT was added. This result suggests that the DDT is able todisplace some of the HBHE. Similarly, when a SSTT-MIP that was templatedfor DDT was challenged 60 ppb DDT, followed by the addition of 85 ppbHBHE, the luminescence dropped in the presence of DDT and then increasedby 122±4% once the HBHE was added. This result suggests that the HBHE isable to displace some of the DDT. Together, these results indicate thatSSTT-MIP strategy can be used for producing luminescence -based sensorsthat are analyte selective.

EXAMPLE 3

This example elucidates some of the SSTT-MIP operational principles. Thesteady-state luminescence anisotropy (r) and multifrequencyphase-modulation traces (equivalent to the time-resolved luminescenceintensity decay) of the SSTT-MIP films (immersed in THF) with andwithout DDT or HBHE were recorded. The results are summarized in Table2. TABLE 2 Recovered steady-state luminescence anisotropies (r) andexcited-state luminescence lifetimes for a DDT responsive SSTT-MTP.Sample ΔF (%)^(a) r τ (ns)/w (ns)^(b) SSTT-MIP 0 0.10 ± 0.01 2.6 ±0.06/1.2 ± 0.2 SSTT-MIP + DDT −27 0.36 ± 0.01 2.6 ± 0.1/0.1 ± 0.1SSTT-MIP + HBHE +120 0.35 ± 0.01 3.4 ± 0.03/0.1 ± 0.1^(a)From FIG. 1 (100 ppb analyte).^(b)The excited-state luminescence lifetimes are reported in terms of aunimodal Lorentzian lifetime distribution where τ is the meanexcited-state lifetime and w is the full width at half maximum for thedistribution.

The Table reveals several important features. First, the Lissamine™rhodamine B (LRB) residue's luminescence anisotropy is much smaller forthe SSTT-MIP without analyte in comparison to the SSTT-MIP with analyte.Second, the intensity decay kinetics for the SSTT-MIP without analyte isclearly distributed, suggesting the luminophore (i.e., LRB) isencountering a distribution of microenvironments/cybotactic regionswithin the SSTT-MIP template site in the absence of analyte. Third, whenDDT is added to the SSTT-MIP the mean LRB lifetime does not change.Fourth, when HBHE is added to the SSTT-MIP the mean LRB lifetimeincreases. Finally, the presence of analyte in the SSTT-MIP causes thelifetime to become single exponential. Together these results indicatethe following: The luminescent report group within the SSTT-MIP templatesite without analyte is relatively mobile and it encounters a broaddistribution of microenvironments/cybotactic regions. In the presence ofDDT, the DDT molecules impede the luminophore's motion within thetemplated site, DDT forces the luminophore into a more discretemicroenvironment or limits access to the other microenvironments, andthe DDT quenches the luminophore fluorescence in a static manner (meanlifetime does not appear to change). In the presence of HBHE, the HBHEmolecules impede the luminophore motion, HBHE forces the luminophoreinto a more discrete microenvironment, and the HBHE protects theluminophore from quenchers and/or increases the local microenvironmentrigidity which causes an increase in the mean lifetime.

The FIG. 3 inset shows the response profile of a SSTT-MIP sensor filmthat was templated for DDT to repeated challenges by 150 ppb DDT. Thesecontinuous flow experiments were performed in a flowing THF stream byinjecting 150 ppb plugs of DDT. The response time (time to reach 90% ofthe maximum signal change) for these 550±150 nm thick SSTT-MIP films ison the order of 20s and the response is reversible to within 4% (20cycles).

In FIG. 4 we summarize a series of response experiments for threedifferent SSTT-MIP sensor films that were each templated for DDT. Theonly difference between these three SSTT-MIP films was the choice of theprecursor ((EtO)₃—Si—(CH₂)₂—Si—(OEt)₃, (EtO)₃—Si-phenyl-Si-(OEt)₃, or(EtO)₃—Si-biphenyl-Si—(OEt)₃) that we used to form the final xerogel.Inspection of these data show several interesting features. First, theseSSTT-MIP sensors are selective for DDT over a number of structurallysimilar analytes. Second, the R′ group choice (FIG. 1) can be used totune the overall sensor selectivity. Specifically, as the size of R′decreases, the overall DDT selectivity improves. These resultsdemonstrate a strategy for forming arrays of SSTT-MIP-based sensorelements wherein each sensor element can be designed for a givenanalyte, and the collective response of the sensor elements for saidanalyte tailored. In this way multiple sensors can be designed for thedetection of the same analyte; a redundant strategy that is selfchecking.

EXAMPLE 4

This embodiment describes the SSMT-MIPs for the detection of two otheranalytes, namely codeine and digitoxin (morphine and digoxin,respectively, served to form the SSMTs). Table 3 shows the effect ofvariables such as xerogel composition, connecting moiety length andluminophore identity for a pair of SSTT-MIP-based sensors designed forthe detection of codeine and digitoxin. TABLE 3 Effects of xerogelcomposition, reporter, tether chemistry and length on analyte detectionlimits. Connec- ting Detection Analyte Xerogel^(a) Reporter Pair^(b)Moiety^(c) Limits Codeine d Dansyl NH₂/SO₂Cl D 25 nM d Dansyl NH₂/SO₂ClC₃ 90 nM d Dansyl NH₂/SO₂Cl C₆ 700 nM d NBD NH₂/SO₂Cl D 1.2 μM e NBDNH₂/SO₂Cl D 1.2 nM Digitoxin d Dansyl NH₂/SO₂Cl D 100 μM f DansylNH₂/SO₂Cl D 1.7 nM g Dansyl NH₂/SO₂Cl D 1.9 nM h NBD NH₂/SO₂Cl D 78 nM^(a)Molar composition of the xerogel.^(b)Function group within the template site/functional bonding group.^(c)D-direct attachment of the bonding group to the luminophore,C₃-propyl spacer between the bonding group and luminophore, C₆-hexylspacer between the bonding group and luminophore.d 45 mol % tetraethylorthosilane (TEOS), 5 mol % octyltrimethoxysilane(OTS), and 50 mol % bis (2-hydroxy-ethyl) aminopropyltriethoxysilane(HAPTS).e 85 mol % TEOS, 5 mol % OTS, and 10 mol % HAPTS.f 47 mol % TEOS, 33 mol % 3,3,3-trifluorpropyltrimethoxysilane(TFP-TMOS), and 20 mole % OTS.g 25 mole % TEOS, 40 mol % (pentafluorophenyl)-propyltrimethoxysilane(PFP-TMOS), and 35 mole % TFP-TMOS.h 50 mole % TEOS, 10 mol % PFP-TMOS, 10 mole % TFP-TMOS, 20 mole % OTS,and 10 mole % TFP-TMOS.

While specific embodiments have been presented in this description,those skilled in the art will recognize that routine modifications canbe made by those skilled in the art without departing from the scope ofthe invention.

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1. A molecularly imprinted polymer for detecting the presence of ananalyte comprising: at least one templated site which is capable ofselectively binding said analyte, said templated site bearing exposedreactive groups, wherein the number of exposed reactive groups exceedsthe number of exposed reactive groups needed to selectively bind theanalyte.
 2. The molecularly imprinted polymer of claim 1 furthercomprising a reporter molecule bound to an exposed reactive group at thetemplated site which is not needed to selectively bind the analyte tothe templated site.
 3. The molecularly imprinted polymer of claim 1,wherein the number of exposed reactive sites at the templated siteexceeds the number of reactive groups needed to selectively bind theanalyte by one. 4 The molecularly imprinted polymer as in claim 1,further comprising an analyte molecule bonded to said one or moreexposed reactive groups.
 5. The molecularly imprinted polymer as inclaim 1, wherein said analyte is a compound selected from the groupcomprised of pharmaceuticals, prostaglandins, steroids, glycosides,deoxyribonucleosides and deoxyribonucleotides.
 6. The molecularlyimprinted polymer as in claim 1 wherein said reporter comprises aluminophore or a chromophore, a connecting group and a binding group. 7.The molecularly imprinted polymer of claim 6, wherein the luminophore isselected from the group consisting of dansyl, fluorescein, BODIPY,rhodamine, tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II)([Ru(dpp)₃]²⁺ and quantum dots.
 8. The molecularly imprinted polymer ofclaim 6, wherein the chromophore is selected from the group consistingof 4-nitroaniline; 2,6-diphenyl-4-(2,4,6-triphenyl-1-pyridinio)phenolate(Reichardt's dye 30);2,6-dichloro-4-(2,4,6-triphenyl-1-pyridinio)phenolate; andN,N-diethyl-4-nitroaniline.
 9. The molecularly imprinted polymer ofclaim 6, wherein the connecting group is a methylene chain, ether chain,polydimethylsiloxane chain, polystyrene chain or amino acid chain. 10.The molecularly imprinted polymer of claim 6, wherein the binding groupis selected from the group consisting of isothiocyanates, succinimidylesters, carboxylic esters, tetrafluorophenyl esters, carbonyl azides,sulfonyl chlorides, arylating agents, aldehydes, iodoacetamides,maleimides, alkyl halides, disulfides, dichlorotriazines,N-methylisatoic anhydride, aminophenylboronic acids, isocyanatesprepared from acyl azides, acyl nitriles, hydrazines, hydroxylaminesamines, carbodiimides, esterification reagents, diazoalkanes, alkylhalides and trifluoromethanesulfonates
 11. The molecularly imprintedpolymer of claim 5, wherein the pharmaceuticals are selected from thegroup consisting of pheytoin, codeine, heroin, cocaine, Δ9-THC,tetracycline, morphine and the SSMT is respectively selected from thegroup consisting of 5-(p-Hydroxyphenyl) or -5-phenylhydantoin, morphine,3-acetylmorphine, p-hydroxycocaine, 8-α-hydroxy-A9-THC, oxytetracyclineand nalbuphine
 12. The molecularly imprinted polymer of claim 5, whereinthe prostaglandins are selected from the group consisting ofprostaglandin A2, prostaglandin B2, prostaglandin E1, prostaglandin E2,prostaglandin F1a, prostaglandin F2a, prostaglandin F2a, and the SSMT isrespectively selected from the group consisting of 19(R)-hydroxyprostaglandin A2, 1 9(R)-Hydroxyprostaglandin B2,19(R)-hydroxyprostaglandin E1, 19(R)-hydroxyprostaglandin E2,19(R)-hydroxyprostaglandin F1a, 19(R)-hydroxyprostaglandin F2a and19(R)-hydroxyprostaglandin F2a.
 13. The molecularly imprinted polymer ofclaim 5, wherein the steroids are selected from the group consisting oftestosterone, estradiol, progesterone, cholesterol and the SSMT isrespectively selected from the group consisting of4-hydroxytestosterone, 4-hydroxyestradiol, 17-c-hydroxyprogesterone and22(r)-hydroxycholesterol.
 14. The molecularly imprinted polymer of claim5, wherein the glycosides are selected from the group consisting ofmethyl α-D-glucopyrnoside, methyl β-D-glucopymoside, digitoxin,2-deoxyribose and the SSMT is respectively selected from the groupconsisting of α-D-glucopyranose, β-D-glucopyranose, digoxin and ribose.15. A method of preparing a site selectively tagged molecularlyimprinted polymer for specifically detecting an analyte comprising thesteps of: a) combining unpolymerized polymer components with one or moresacrificial spacer molecular template (SSMT) molecules, wherein the SSMTmolecule has at least one more reactive groups than the analyte; b)allowing polymerization of the unpolymerized components thereby formingtemplated sites in the polymer, wherein each templated site is bonded toa SSMT molecules via a plurality of bonds; c) releasing the SSMTmolecules from the templated sites thereby forming templated siteshaving a plurality of exposed reactive groups such that the number ofexposed reactive groups at each templated site is greater than thenumber of reactive groups on the analyte molecule; d) effecting thebinding of the analyte molecule to the templated site having a pluralityof exposed reactive groups, wherein at least one exposed reactive groupat each templated site is left free; e) effecting the binding of areporter molecule to the free exposed reactive group; and f) releasingthe analyte molecule thereby providing a site selectively taggedmolecularly imprinted polymer which can specifically detect the presenceof the analyte in a test sample.
 16. The method of claim 15, wherein thepolymer is a xerogel or an aerogel.
 17. The method as in claim 15,wherein the at least one more reactive groups on the SSMT moleculecomprises a hydroxyl group.
 18. The method as in claim 15, wherein thewherein the unpolymerized components in step a) are combined with morethan one type of SSMT molecule with each type of SSMT molecule being amimic for the same analyte thereby forming site selectively taggedmolecularly imprinted polymer having templated sites with differentdetection limits for the same analyte.
 19. The method as in claim 15wherein step a) wherein the SSMT which is used to form the templatedsite is removed without leaving chemical residues.
 20. The method as inclaim 19 wherein the SSMT is released from the templated site in step c)without leaving chemical residues on the template site.
 21. The methodas in claim 20, wherein the residues are amino groups.
 22. The method asin claim 15 wherein said reporter comprises a luminophore or achromophore, a connecting group and a binding group.
 23. The method ofclaim 15, wherein the reporter is a luminophore selected from the groupconsisting of dansyl, fluorescein, BODIPY, rhodamine,tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) ([Ru(dpp)₃]²⁺ andquantum dots
 24. The method of claim 15, wherein the reporter is achromophore is selected from the group consisting of 4-nitroaniline;2,6-diphenyl-4-(2,4,6-triphenyl-1-pyridinio)phenolate (Reichardt's dye30); 2,6-dichloro-4-(2,4,6-triphenyl-1-pyridinio)phenolate; andN,N-diethyl-4-nitroaniline
 25. The method of claim 22, wherein theconnecting group is selected from the group consisting of a methylenechain, ether chain, polydimethylsiloxane chain, polystyrene chain andamino acid chain.
 26. The method of claim 22, wherein the binding groupis selected from the group consisting of isothiocyanates, succinimidylesters, carboxylic esters, tetrafluorophenyl esters, carbonyl azides,sulfonyl chlorides, arylating agents, aldehydes, iodoacetamides,maleimides, alkyl halides, disulfides, dichlorotriazines,N-methylisatoic anhydride, aminophenylboronic acids, isocyanatesprepared from acyl azides, acyl nitriles, hydrazines, hydroxylaminesamines, carbodiimides, esterification reagents, diazoalkanes, alkylhalides and trifluoromethanesulfonates.
 27. The method of claim 15,wherein the analyte is a compound selected from the group comprised ofpharmaceuticals, prostaglandins, steroids, glycosides,deoxyribonucleosides and deoxyribonucleotides.
 28. The method of claim27, wherein the pharmaceuticals are selected from the group consistingof pheytoin, codeine, heroin, cocaine, Δ9-THC, tetracycline, morphineand the SSMT is respectively selected from the group consisting of5-(p-hydroxyphenyl) or -5-phenylhydantoin, morphine,3-acetylmorphine,p-hydroxycocaine, 8-α-hydroxy-Δ9-THC, oxytetracyclineand nalbuphine
 28. The method of claim 15, wherein the prostaglandinsare selected from the group consisting of prostaglandin A2,prostaglandin B2, prostaglandin E1, prostaglandin E2, prostaglandin F1a,prostaglandin F2a, prostaglandin F2a, and the SSMT is respectivelyselected from the group consisting of 19(R)-hydroxyprostaglandin A2,19(R)-hydroxyprostaglandin B2, 19(R)-hydroxyprostaglandin E1,19(R)-hydroxyprostaglandin E2, 19(R)-hydroxyprostaglandin F1a,19(R)-hydroxyprostaglandin F2a and 19(R)-hydroxyprostaglandin F2a. 29.The method of claim 15, wherein the steroids are selected from the groupconsisting of testosterone, estradiol, progesterone, cholesterol and theSSMT is respectively selected from the group consisting of4-hydroxytestosterone, 4-hydroxyestradiol, 17-α-hydroxyprogesterone and22(r)-hydroxycholesterol.
 30. The method of claim 15, wherein theglycosides are selected from the group consisting of methylα-D-glucopyrnoside, methyl β-D-glucopyrnoside, digitoxin, 2-deoxyriboseand the SSMT is respectively selected from the group consisting ofα-D-glucopyranose, β-D-glucopyranose, digoxin and ribose.
 31. A methodfor detecting the presence of an analyte in a test sample comprising thesteps of: a) contacting the test sample with a site selectively taggedmolecularly imprinted polymer which has at least one template site whichis specific for the analyte and wherein the templated site has at leastone reporter molecule bonded to it; b) detecting a change in theemission from the reporter molecule upon exposure to the test sample,wherein a change in the emission from reporter molecule indicates thepresence of the analyte in the test sample.